Method for detecting genes sensitive to low-level ionizing radiation

ABSTRACT

A method for detecting genes sensitive to low-level ionizing radiation and genes detected by the method. More specifically, genes sensitive to low-level ionizing radiation, discovered in a carcinogenic entity and verified in a normal entity are detected by subjecting a cancerous AKR/J mouse and a normal ICR mouse to low-level radiation. Thymus is collected therefrom, and glycometabolism-related genes are classified via microarray processing of the thymus. The genes are amplified and the levels of gene expression are measured. Thus, a gene having a specific reaction to radiation can be accurately detected by preventing the interference of confounding variables.

RELATED APPLICATIONS

This application is a §371 application from PCT/KR2012/003928 filed May18, 2012, which claims priority from Korean Patent Application No.10-2012-0049544 filed May 10, 2012, each of which is herein incorporatedby reference in its entirety.

TECHNICAL FIELD

The present invention relates to a method for detecting genes sensitiveto a low level of ionizing radiation and genes detected by the method,and more particularly, to a method for detecting genes sensitive to alow level of ionizing radiation, the method including irradiatingcancer-induced mice and normal mice with a low level of radiation, andscreening glucose metabolism-related genes, which are observed commonlyin the normal mice and the cancer-induced mice, from the thymi of themice.

BACKGROUND ART

With an increase in the industrial and medical use of radiation, variousstudies on the effects of radiation on the human body have beenconducted, and particularly, cancer therapy with radiation has receivedattention. It is known that high doses of ionizing radiation cause DNAdamage, genetic modification, and diseases, including cancer, but aradiation dose of 200 mGy or less and a radiation dose rate of 6 mGy/hror less inhibit cancer development by activating immune responses.

In general, studies on the relationship between radiation and cancerdevelopment, particularly gene responses to radiation, have beenconducted, but confounding factors have significantly affected theresults to reduce the reliability of the results. However, most studiesconducted to date could not explain various responses, which occur inthe cells, tissues and organs of the body in the body stage, becausethese studies were performed using gene-modified cell lines or cancercell lines. In other words, because gene responses were evaluated usinggeneral mice, a variety of genes were expressed, and because cancerdevelopment was not limited to a specific organ, it was difficult toanalyze gene responses.

In prior art methods that use cells for cancer research, genes weremodified, or cancer cells lacking p53 that is important in cancerdevelopment were irradiated. For this reason, there was a problem inthat the results could not be applied to individuals, because they didfundamentally differ from the responses of normal cells. To overcomethis problem, studies on the effects of radiation on cancer developmenthave been conducted using mice having a gene similarity of 95% or morewith humans. However, cancer incidence in general mice is very low, andthus a variety of mouse models for cancer research have been used.

In prior studies, a variety of methods were used to screen glucosemetabolism-related genes sensitive to a low level (0.7 mGy/hr) ofradiation. However, genes disclosed in the present invention are not yetknown as genes sensitive to a low level (0.7 mGy/hr) of radiation.Technologies prior to the identification of the profile of genesaccording to the present invention are as follows.

(1) Cancer cells are characterized by activating glucose uptake andglycolysis (Warburg O, Science 1956; 123: 309-314).

(2) Activated glucose metabolism inhibited p53 activity in thymus,inhibited puma induction, affected the balance between the expression ofBcl2 family proteins and the inhibition of apoptosis, and maintainedcancer survival (Zhao Y et. al., J Biol Chem 2008; 283: 36344-36353).

(3) Apoptosis increased in the ileum of Akt1 knockdown mice irradiatedwith ionizing radiation (Plastaras et al., 2008).

Accordingly, the present inventors have identified the profile ofglucose metabolism-related genes sensitive to a low level of ionizingradiation.

DISCLOSURE Technical Problem

It is an object of the present invention to a method for detecting agene sensitive to a low level of ionizing radiation, and a gene detectedby the method.

Technical Solution

In order to accomplish the above objects, the present invention providesa method for detecting a gene that is sensitive to a low level ofionizing radiation and is identified in a cancer-induced individual andverified using a normal individual, the method including the steps of:I) irradiating an AKR/J mouse and an ICR mouse with a low level ofradiation; II) extracting thymi from the AKR/J mice and the ICR mice;III) subjecting the thymi to microarray analysis; IV) selecting aglucose metabolism-related gene from the microarray analysis; and V)amplifying the gene and measuring the expression level of the gene.

The present invention also provides a marker for diagnosing aradiation-sensitive or radiation-induced cancer, the marker includingthe nucleotide sequence of a glucose metabolism-related gene selectedfrom the group consisting of IRS1 (NM 010570), Glut1 (MM 011400), Glut4(NM 009204), LPK (NM 013631) and G6pc (NM 008061), which are involved inworsening of thymic cancer.

The present invention also provides a kit for diagnosing aradiation-sensitive or radiation-induced cancer, the kit including theabove marker.

The present invention also provides a microarray for diagnosing aradiation-sensitive or radiation-induced cancer, the microarrayincluding the above marker.

The present invention also provides a method for detecting a genecapable of measuring a radiation-sensitive or radiation-induced cancer,the method including the steps of: I) irradiating a mammal having thymiccancer with radiation; II) bringing a test substance into contact with athymic tissue extracted from the irradiated mammal; and III) measuring,from the thymic tissue, a change in the expression of a glucosemetabolism-related gene selected from the group consisting of IRS1(NM_010570), Glut1 (MM_011400), Glut4 (NM_009204), LPK (NM_013631) andG6pc (NM_008061), which are involved in worsening of thymic cancer.

Hereinafter, the present invention will be described in detail.

Many studies on the effects of radiation on cancer development among theeffects of radiation on the human body have been conducted, but it wasdifficult to explain various responses of the body to radiation(responses of genes to radiation), because these studies were performedusing cancer cells, gene-modified cell lines or general mice.Particularly, the profile of glucose metabolism-related genes sensitiveto ionizing radiation in individuals has not yet been identified, andthe functions of these genes have not been explained. Accordingly, thepresent invention is intended to (1) identify the profile of glucosemetabolism-related genes that are expressed specifically in thymus andsensitive to a low level of radiation, and analyze the functions of thegenes, after irradiating a low level (0.7 mGy/hr) of radiation (cancerdevelopment stimulator) to normal ICR mice and AKR/J mice that developthymic cancer, and 2) diagnose the stage of development of thymic cancerusing the profile of glucose metabolism-related genes.

The present invention provides a method for detecting a gene that issensitive to a low level of ionizing radiation and is identified in acancer-induced individual and verified using a normal individual, themethod including the steps of: I) irradiating an AKR/J mouse and an ICRmouse with a low level of radiation; II) extracting thymi from the AKR/Jmice and the ICR mice; III) subjecting the thymi to microarray analysis;IV) selecting a glucose metabolism-related gene from the microarrayanalysis; and V) amplifying the gene and measuring the expression levelof the gene.

In the inventive method for detecting a gene sensitive to a low level ofionizing radiation, irradiating the mouse with the low level ofradiation is preferably performed by irradiating gamma radiation(Cs-137) at a dose rate of 0.7 mGy/hr to a final dose of 1.7 Gy. Themethod according to the present invention is preferably used forpreparation of a kit for diagnosing thymic cancer, evaluation of thedegrees of progression and treatment of cancer in a cancer patient,evaluation of the relationship between radiation exposure of industrialand medical workers and cancer development, evaluation of the causalrelation between radiation and cancer development, biological evaluationof radiation exposure dose, or evaluation of the degrees of developmentand progression of thymic cancer caused by a low level of radiation.

In addition, the inventive method for detecting a gene sensitive to alow level of ionizing radiation, the cancer is preferably thymic cancer,and extracting the thymi in step II) is preferably performed at a timepoint when the mouse starts to die of the cancer.

Furthermore, in the inventive method for detecting a gene sensitive to alow level of ionizing radiation, the glucose metabolism-related gene ispreferably selected from the group consisting of IRS1 (NM_010570), Glut1(MM_011400), Glut4 (NM_009204), LPK (NM_013631) and G6pc (NM_008061).Preferably, the IRS1 (NM_010570) is amplified using primers havingsequences set forth in SEQ ID NOS: 1 and 2; the Glut1 (MM_011400) geneis amplified using primers having sequences set forth in SEQ ID NOS: 3and 4; the Glut4 (NM_009204) gene is amplified using primers havingsequences set forth in SEQ ID NOS: 5 and 6; the LPK (NM_013631) gene isamplified using primers having sequences set forth in SEQ ID NOS: 7 and8; and the G6pc (NM_008061) gene is amplified using primers havingsequences set forth in SEQ ID NOS: 9 and 10.

In step IV) of selecting the glucose metabolism-related gene from themicroarray analysis, a gene overexpressed or underexpressed in thecancer-induced individual after irradiation compared to in thecancer-induced individual before irradiation is detected by microarrayanalysis, and then verified using primers having sequences of SEQ IDNOS: 1 to 10, and the overexpressed or underexpressed gene is identifiedby performing a search for the function thereof. The microarray analysisis described in the Examples below, and a search for the function of thegene was performed in the Examples through the DAVID bioinformaticsdatabase and (apps1.niaid.nih.gov) and the PubMed database(www.ncbi.nlm.nih.gov), but is not limited thereto.

As used herein, “gene sensitive to a low level of radiation” refers to agene that is differentially overexpressed or underexpressed in acancer-induced individual after radiation compared to beforeirradiation. In other words, the gene refers to a gene whose expressionpattern is changed by stimulation with radiation, and it may be a targetgene associated with a specific cancer, that is, an oncogene or a tumorsuppressor gene. When this cancer-specific gene is detected, a molecularmechanism for radiotherapy of cancer patients can be established, whichcan contribute to an increase in the effect of radiotherapy, and aplatform for the development of agents or methods for treating cancer atthe biomolecular level can be provided by screening novel oncogenes ortumor suppressor genes and regulating the expression thereof.

The present invention also provides a marker for diagnosing aradiation-sensitive or radiation-induced cancer, the marker includingthe nucleotide sequence of a glucose metabolism-related gene selectedfrom the group consisting of IRS1 (NM_010570), Glut1 (MM_011400), Glut4(NM_009204), LPK (NM_013631) and G6pc (NM_008061), which are involved inworsening of thymic cancer.

The present invention also provides a kit for diagnosing aradiation-sensitive or radiation-induced cancer, the kit including theabove marker.

The present invention also provides a microarray for diagnosing aradiation-sensitive or radiation-induced cancer, the microarrayincluding the above marker.

The present invention also provides a method for detecting a genecapable of measuring a radiation-sensitive or radiation-induced cancer,the method including the steps of: I) irradiating a mammal having thymiccancer with radiation; II) bringing a test substance into contact with athymic tissue extracted from the irradiated mammal; and III) measuring,from the thymic tissue, a change in the expression of a glucosemetabolism-related gene selected from the group consisting of IRS1(NM_010570), Glut1 (MM_011400), Glut4 (NM_009204), LPK (NM_013631) andG6pc (NM_008061), which are involved in worsening of thymic cancer.

In the present invention, AKR/J mice (models for thymic cancer research)and healthy ICR mice were irradiated with a low level (0.7 mGy/hr) ofgamma radiation (Cs-137), and thymi were extracted at a time point (day100) when the AKR/J mice started to die of thymic cancer. The extractedthymi were analyzed by microarray analysis, and then glucosemetabolism-related genes that responded sensitively to the low level ofradiation (0.7 mGy/hr) were selected through the DAVID bioinformaticsdatabase, and subjected to nucleic acid amplification, and theexpression levels thereof were measured.

As a result, five genes (IRS1, Glut1, Glut4, LPK and G6pc), whichresponded sensitively to the low level of radiation (0.7 mGy/hr) and areimportant in glucose metabolism, were screened in the present invention,and the functions of the glucose metabolism-related genes (IRS1, Glut1,Glut4, LPK and G6pc) that responded sensitively to the low level ofradiation (0.7 mGy/hr) were elucidated. In addition, the glucosemetabolism-related genes that responded sensitively to the low level ofradiation could be consistently observed by extracting thymi at day 100when death caused by thymic cancer was observed.

Therefore, the present invention may be used to: (1) identify theprofile of genes for development of a kit for diagnosing thymic cancer;(2) identify a marker for evaluating the relation of cause and effect ofcancer development in industrial and medical workers who live inenvironments having a low level of radiation; (3) identify the profileof genes for information, which enable the diagnosis of cancerdevelopment in cancer patients and allow a cancer therapeutic method tobe established; (4) identify a marker for evaluating the causal relationbetween radiation exposure and the development of thymic cancer; 5)identify a novel gene marker that may be widely used for biologicalevaluation of a low level of radiation exposure; and (6) understandionizing radiation-sensitive glucose metabolism signaling that may beused as a target therapy for a low level of radiation exposure.

Advantageous Effects

The method for detecting a gene sensitive to a low level of ionizingradiation as described above may be used to establish the profile ofglucose metabolism-related marker genes sensitive to a low level (0.7mGy/hr) of radiation in order to prepare a kit for diagnosing thymiccancer, and may provide a glucose metabolism-related marker genesensitive to a low level (0.7 mGy/hr) of radiation, which can be used toevaluate the degrees of progression and progression of cancer in cancerpatients. Also, the method according to the present invention mayprovide a glucose metabolism-related marker gene sensitive to a lowlevel (0.7 mGy/hr) of radiation, which can be used to evaluate therelationship between the radiation exposure of industrial and medicalworkers and cancer development. Further, it may provide a glucosemetabolism-related marker sensitive to a low level of radiation, whichcan be used to evaluate the causal relation between radiation and cancerdevelopment. In addition, it may provide a novel marker that can be usedfor biological evaluation of radiation exposure dose. Also, it mayprovide a glucose metabolism-related marker that can be used to evaluatethe degrees of development and progression of thymic cancer caused by alow level (0.7 mGy/hr) of radiation, and the effect of low-degreeradiation on the suppression of thymic cancer.

DESCRIPTION OF DRAWINGS

FIG. 1 schematically shows the functions of glucose metabolism-relatedgenes that suppress thymic cancer upon irradiation with a low level (0.7mGy/hr) of radiation.

FIG. 2 is a graph showing the results obtained by irradiating AKR/J micewith a low level (0.7 mGy/hr) of radiation, and measuring the weight ofthymi of the mice at a time point (day 100) when the mice started to dieof thymic cancer during their housing, in order to analyze the responsesof glucose metabolism-related genes sensitive to radiation based on thethymus weight.

MODE FOR INVENTION

Hereinafter, the present invention will be described in further detailwith reference to examples. It is to be understood, however, that theseexamples are for illustrative purposes only and are not intended tolimit the scope of the present invention.

Example 1

6-Week-old female AKR/J mice (models for thymic cancer research) and6-week-old female ICR mice were purchased from SLC Co., Ltd. (Japan). Alow level of radiation (¹³⁷Cs) was irradiated to the AKR/J mice using agamma-ray generator (IBL 147C, CIS bio international, France) at a doserate of 0.7 mGy/hr) so as to reach a final dose of 4.5 Gy. Aftercompletion of irradiation with the low level of radiation, the mice weretransferred into a sterilized housing system shielded from radiation,and were housed therein for 100 days while the observation ofdevelopment of thymic cancer was performed. For gene analysis, under thesame experimental conditions, normal mice (ICR mice) housed separatelyfrom the AKR/J mice were irradiated with a low level of radiation (0.7mGy/hr). After 100 days, thymi were extracted from the mice and frozenrapidly in liquid nitrogen, after which gene analysis was performed.

To confirm the results, the genes were subjected to nucleic acidamplification. Specifically, the thymi extracted from the AKR/J and ICRmice irradiated with the low level of radiation (0.7 mGy/hr) wereanalyzed by a microarray, and glucose metabolism-related genes thatresponded sensitively to the low level of radiation were amplified usingthe primers shown in Table 1 below in order to measure the expressionlevels thereof.

TABLE 1  Gene Forward Reverse Gene No. name (5' --> 3') (5' --> 3')NM_010570 IRS1 GGTGCAGCAGAT GGTCTTCTGA CTGGATAA TGGGAAATGG NM_011400Glutl ATCTTCGAGA ACAAACAGCGA AGGCAGGTGT CACCACAGT NM_009204 Glut4AGAGAGCGTC ACAGCATTGA CAATGTCCTT TGCCTGAGAG NM_013631 LPK TGATCACTAAGGTCTCTCCA GGCTCGACCA GACAGCATGA NM_008061 G6pc TCCTCTTTCC TCCACTTGAACATCTGGTTC GACGAGGTTG

After irradiation of the AKR/J and ICR mice with the low level ofradiation (0.7 mGy/hr), the mice were housed, and thymi were extractedfrom the mice at a time point (day 100) when the AKR/J mice started todie of thymic cancer. The extracted thymi were microarrayed, and glucosemetabolism-related genes that responded sensitively to the low level ofradiation were selected, and then subjected to nucleic acidamplification, and the expression levels thereof were measured. As aresult, it was shown that, in the mice irradiated with the low level ofradiation, glucose metabolism-related genes (IRS1, Glut1, Glut4, LPK andG6pc) responded sensitively to the low level of radiation. The resultsare shown in Table 2 below.

TABLE 2 Quantitative nucleic Microarray amplification AKR/J AKR/J GeneNo. Gene name ICR mice mice ICR mice mice NM_010570 IRS1 0.8 1.6 1.3 ±0.1 1.0 ± 0.2 NM_011400 Glut1 1.0 1.1 2.3 ± 0.5 1.3 ± 0.4 NM_009204Glut4 0.4 1.8 1.2 ± 0.1 1.1 ± 0.1 NM_013631 LPK 1.0 1.4 2.4 ± 0.9 3.9 ±1.9 NM_008061 G6pc 0.8 0.7 1.4 ± 0.4 2.7 ± 0.6 *Expression fold value ±SD

FIG. 1 schematically shows the functions of glucose metabolism-relatedgenes that worsen thymic cancer due to irradiation with a low level ofradiation (0.7 mGy/hr). As can be seen therein, the low level ofradiation reduced the expression of insulin signaling gene (IRS1),glucose uptake genes (Glut1 and Glut4), glycolysis gene (LPK) andgluconeogenesis gene (G6pc), and suppressed thymic cancer.

FIG. 2 shows FIG. 2 is a graph showing the results obtained byirradiating AKR/J mice with a low level (0.7 mGy/hr) of radiation, andmeasuring the weight of thymi of the mice at a time point (day 100) whenthe mice started to die of thymic cancer during their housing, in orderto analyze the responses of glucose metabolism-related genes sensitiveto radiation based on the thymus weight.

According to the present invention, glucose metabolism-related genesthat respond sensitively to a low level of radiation can be consistentlymeasured by extracting thymi in an early stage of cancer development inwhich mice start to die of thymic cancer, and comparing the weights ofthe extracted thymi.

The invention claimed is:
 1. A method for detecting a gene sensitive toan ionizing radiation, the method comprising the steps of: irradiating acancer-induced AKR/J mouse and an ICR mouse with a gamma radiation at adose rate of a dose rate of 0.7 mGy/hr to a final dose of 1.7 Gy;extracting thymi from the AKR/J mouse and the ICR mouse; subjecting thethymi to a microarray analysis; selecting a G6pc gene as a glucosemetabolism-related gene from the microarray analysis; and amplifying theG6pc gene and measuring an expression level of the gene.
 2. The methodof claim 1, wherein the cancer is thymic cancer.
 3. The method of claim1, further comprising the step of extracting the thymi at a time pointwhen the mouse starts to die of the cancer.
 4. The method of claim 1,wherein the step of amplifying the G6pc gene uses primers havingsequences set forth in SEQ ID NOS: 9 and 10.